The present invention relates to a microfluidic system and to a method for sorting cell clusters, such as islets of Langerhans, and for the continuous and automated encapsulation of the clusters, once sorted, in capsules of sizes suitable for those of these sorted clusters. The invention applies in particular to the coupling between sorting and encapsulation of such cell clusters, but also, more generally, of cells, of bacteria, of organelles or of liposomes, in particular.
Cell encapsulation is a technique which consists in immobilizing cells or cell clusters in microcapsules, so as to protect them against attacks by the immune system during transplantation. The porosity of the capsules should allow the entry of low-molecular-weight molecules essential to the metabolism of the encapsulated cells, such as molecules of nutrients, of oxygen, etc., while at the same time preventing the entry of substances of higher molecular weight, such as antibodies or the cells of the immune system. This selective permeability of the capsules is thus designed to ensure the absence of direct contact between the encapsulated cells of the donor and the cells of the immune system of the transplant recipient, thereby making it possible to limit the doses of immunosuppressor treatment used during the transplantation (this treatment having strong side effects).
Among the multiple applications of the encapsulation, mention may be made of that of islets of Langerhans, which are clusters of fragile cells located in the pancreas and consisting of several cell types, including β-cells which regulate glycemia in the body by producing insulin. Encapsulation of these islets is an alternative to the conventional cell therapies (e.g. transplantation of pancreas or of islets) used to treat insulin-dependent diabetes, an autoimmune disease in which the immune system destroys its own insulin-producing β-cells.
The capsules produced should meet certain criteria, including biocompatibility, mechanical strength and selective permeability, in particular. Another essential criterion is the size of the capsules, since, by adjusting the size of the cell clusters as well as possible (see reference [1]):                the amount of “needless” polymer around the cells is reduced, and therefore the response time of the latter is reduced. For example, the regulation of the glycemia by islets of Langerhans encapsulated in capsules of appropriate size will be more rapid, since the glucose will diffuse more rapidly to the islet and the insulin produced will escape therefrom more rapidly;        the viability of the encapsulated islets is maximized due to the fact that the diffusion of oxygen therein is more rapid, thereby improving the oxygenation of the cells and reducing the risks of appearance of necrosed zones; and        the volume of capsules to be transplanted is reduced, which can enable the capsules to be implanted in zones more suitable for tissue revascularization. In fact, this revascularization is essential in order to prevent necrosis of the encapsulated cells, since the cells must be located in proximity to the blood network so as to be well supplied with nutrients and with oxygen, in particular. For example, for the treatment of insulin-dependent diabetes, this reduced volume makes it possible to implant the encapsulated islets in the liver or the spleen, regions which are more favorable to revascularization and the peritoneal cavity where capsules are conventionally implanted for reasons of steric hindrance.        
While the properties of biocompatibility, mechanical strength or selective permeability appear to be well acquired according to the literature, the same cannot be said of the size of the capsules, which is particularly problematic for the encapsulation of islets of Langerhans. This is because, in all the documents known to the applicant to date, the size of the capsules formed around these islets is fixed and on average of the order of 600 to 800 μm, whereas these islets have a size ranging from 20 to 400 μm only. A capsule size which is fixed and identical whatever the size of the islet therefore poses a problem, all the more so since recent studies have shown that the most effective islets are the smallest ones (see reference [2]).
The principal known encapsulation methods use, according to preference:                a coaxial liquid or air jet, the capsules produced having a size ranging between 400 μm and 800 μm (however, the average size of the capsules produced is closer to 600-800 μm than to 400 μm);        a potential difference, which is the encapsulation technique most commonly used when the priority is to reduce the size of the capsules (the size of the capsules ranges, in this case, between 200 and 800 μm); or        a vibration technique, which has the drawback of sometimes being limited by the viscosities of the solutions used.        
The main drawbacks of these techniques are:                the sizes of the capsules, which are not necessarily suitable for those of the islets of Langerhans to be encapsulated;        the lack of automation of the encapsulation procedure, where the capsules are gelled while falling into a bath of polycations and are subsequently recovered manually, which generates a heterogeneity in the polymerization time from one capsule to another;        the size dispersion of the capsules, which increases when the size of the drops decreases; and        a lack of reproducibility of the capsules produced, which are not necessarily spherical.        
Microfluidic systems suitable for size-sorting of bacteria, of cells, of organelles, of viruses, of nucleic acids or even of proteins have recently been developed, and among said systems, mention may be made of:                those which perform sorting by “deterministic lateral displacement” or “DLD” (see references [6-8] and, for example, document WO-A-2004/037374, US-A-2007/0059781 and US-A-2007/0026381), which are based on the use of a periodic array of obstacles which will disturb or not the path of the particles to be sorted. The particles smaller than the critical size Dc, fixed by the geometry of the device, are not, overall, deflected by these obstacles, such as posts, whereas those larger than this size Dc are deflected in the same direction at each row of posts. The path of the largest particles is therefore in the end deflected relative to that of the smallest, thereby enabling the size-separation of the particles, it being specified that, in the DLD technique, the spacing between two adjacent posts is always greater than the size of the particles to be deflected. This device is suitable for blood samples (separation of red blood cells, white blood cells and of the plasma);        systems which perform sorting by hydrodynamic filtration (see references [9, 10] and documents JP-A-2007 021465, JP-A-2006 263693, and JP-A-2004 154747), which consists in adapting the fluidic resistances of transverse channels by choosing an appropriate rate of flow rates between the main channel and these transverse channels. As a result, the particles of which the size is greater than a critical size (fixed by the value of the fluidic resistance) cannot enter into these transverse channels, even if their size is less than the width of the transverse channels;        simpler systems of sorting by size, using only flow line deflection (see references [11, 12] and, for example, document WO-A-2006/102258) where, in the sorting region, the flow lines are deflected toward a low pressure region: the difference in positioning of the flow lines is accentuated, and since the particles follow the flow lines on which their center of inertia is positioned, the difference in position between small and large particles is accentuated;        sorting systems using filters which make it possible either to allow molecules having a size less than a critical value to pass (see document US-A-2005/0133480), or to allow only the fluid to pass, so as to concentrate the particles or separate the fluid which transports them (see, in this case, document WO-A-2006/079007). The principal limitation of these filter-sorting systems is the risk of clogging of the channels by the particles; and        sorting systems where the microfluidic device is coupled to an external field, for instance optical fluorescence or absorbance measurement (see documents WO-A-2002/023163 and WO-A-02/40874), optical traps, dielectrophoresis, conductimetry, potentiometry or amperometry measurements, detection of ligand/receptor binding, etc.        
A major drawback of all the microfluidic sorting systems presented in these documents is that they are not at all suitable for sorting cell clusters, such as islets of Langerhans or other relatively noncohesive clusters of similar sizes. In fact, and as explained previously, each of these clusters behaves quite differently from a cell due to its size (from 20 μm to 400 μm for islets of Langerhans, against about ten μm for a single cell) and also due to its weak cohesion (which means that weak shear stresses must be used in the microfluidic sorting system used).
The only system known to the applicant for sorting such cell clusters is the flow cytometry known as “COPAS” which is marketed by the company Union Biometrica. This system, which is not of the microfluidic type, sorts the clusters by size, by measuring their respective times of flight in the beam of a laser radiation (see reference [13]).
Microfluidic encapsulation systems have also recently been developed, which use emulsions that can in particular be formed:                at a T-junction (see reference [14]),        at the orifice of a microfluidic flow focusing device, MFFD (see reference [15]),        through structured microchannels (cf. reference [16]), or        through nozzles (see reference [17]).        
These encapsulation systems are the subject of numerous documents, among which are the documents WO-A-2004/071638, US-A-2007/0054119, FR-A-2776535, JP-A-2003 071261 and US-A-2006/0121122 and, more particularly for the encapsulation of cells or cell clusters and the gelling of the capsules formed, the documents US-A-2006/0051329, WO-A-2005/103106 and WO-A-2006/078841.
The gelling step is carried out directly on the microsystem with microchannels in the form of a coil or H-shaped microchannels, as described in documents US-A-2006/0051329 and WO-A-2005/103106.
The principal drawback of these microfluidic encapsulation systems is the same as that mentioned above in the introduction, which is the fact that a single capsule size is obtained whatever the size of the cell clusters. To the applicant's knowledge, only the device of Wyman et al. (see reference [18] and document US-A-2007/0009668) makes it possible to adapt the size of the capsule to the size of the cell clusters, such as islets of Langerhans, by enveloping them in capsules which have a constant thickness in the region of 20 μm, but independently of the size of the islets encapsulated. In the latter document, an aqueous phase is placed above an oil phase and, by adjusting the respective relative densities of these two phases, the islets are found at the water/oil interface. A sampling tube placed in the oil at a certain distance from the interface makes it possible to draw off the aqueous phase and the islets in a fine jet, which, under the effect of the surface tension, breaks up, leaving at the surface of the islets a fine coating of hydrogel of fixed thickness, which is polymerized by UV irradiation. This device is, however, a macroscopic device, and not a microfluidic system.